Design, Synthesis, and Biological Evaluation of Phenyloxadiazole Sulfoxide Derivatives as Potent Pseudomonas aeruginosa Biofilm Inhibitors

With the development of antimicrobial agents, researchers have developed new strategies through key regulatory systems to block the expression of virulence genes without affecting bacterial growth. This strategy can minimize the selective pressure that leads to the emergence of resistance. Quorum sensing (QS) is an intercellular communication system that plays a key role in the regulation of bacterial virulence and biofilm formation. Studies have revealed that the QS system controls 4–6% of the total number of P. aeruginosa genes, and quorum sensing inhibitors (QSIs) could be a promising target for developing new prevention and treatment strategies against P. aeruginosa infection. In this study, four series of phenyloxadiazole and phenyltetrazole sulfoxide derivatives were synthesized and evaluated for their inhibitory effects on P. aeruginosa PAO1 biofilm formation. Our results showed that 5b had biofilm inhibitory activity and reduced the production of QS-regulated virulence factors in P. aeruginosa. In addition, silico molecular docking studies have shown that 5b binds to the P. aeruginosa QS receptor protein LasR through hydrogen bond interaction. Preliminary structure–activity relationship and docking studies show that 5b has broad application prospects as an anti-biofilm compound, and further research will be carried out in the future to solve the problem of microbial resistance.


Introduction
As a common Gram-negative bacterium, Pseudomonas aeruginosa (P. aeruginosa or PA) is one of the main pathogens causing nosocomial infections and ventilator-associated pneumonia. It mainly affects patients that are immunocompromised, such as those with severe burns, chemotherapy, or HIV etc. [1-3] The ability of P. aeruginosa to form antibiotic-resistant biofilms is a significant factor for its notorious persistence in clinical settings [4]. It has been estimated that over 80% of hospital-acquired infections are biofilm-mediated, and that treatment of these biofilm-based infections costs more than USD 1 billion annually [5,6]. The biofilms' surface-associated bacteria are microbial communities that attach to inert or living surfaces and encase them in a self-produced extracellular polymeric matrix [7]. Biofilm matrices usually contain polysaccharides, water, proteins, and extracellular DNA, but their composition varies according to bacterial species and environmental conditions [8][9][10]. The bacteria in biofilms are more tolerant to conventional antibiotics, and they are protected from environmental stress and host immune response [11]. P. aeruginosa forms biofilm on many surfaces, including cystic fibrosis-affected tissues in lungs and on medicinal implants, such as catheters, artificial hips, and contact lenses, which can cause severe infections that Hence, in this study, four series of phenyloxadiazole and phenyltetrazole sulfoxide derivatives were designed and synthesized to optimize potent P. aeruginosa QSIs. Through the biofilm inhibitory behavior of P. aeruginosa PAO1 and the fluorescence expression analysis of reporter strains (PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp), 5b was found to be the most active biofilm inhibitor. We also tested 5b for QS-activated virulence factors (elastase, rhamnolipid, and pyocyanin) production. Finally, we explored the binding effects between 5b and the LasR receptor protein with molecular docking.

Chemistry
A series of phenyldioxazole and phenyltetrazole sulfoxide derivatives were synthesized. The preparation methods for the titled compounds are described in Scheme 1. The different substituted benzyl bromides 2 were added dropwise to a solution of phenyldioxazole thiol or phenyltetrazole thiol 1 and Et3N in MeCN at room temperature to obtain intermediate thioethers 3. Then, intermediates 3 were oxidized by m-chloroperoxybenzoic acid (mCPBA) in dichloromethane to obtain title compounds 4a-4o (containing phenyltetrazole), 5a-5o (containing phenyldioxazole), 6a-6o (containing 4-methoxylphenyltetrazole), and 7a-7o (containing 4-chlorophenyldioxazole) [37]. 1 H NMR, 13 C NMR spectra and HRMS spectra of all compounds are shown in the experimental section and supplementary materials.

Chemistry
A series of phenyldioxazole and phenyltetrazole sulfoxide derivatives were synthesized. The preparation methods for the titled compounds are described in Scheme 1. The different substituted benzyl bromides 2 were added dropwise to a solution of phenyldioxazole thiol or phenyltetrazole thiol 1 and Et 3 N in MeCN at room temperature to obtain intermediate thioethers 3. Then, intermediates 3 were oxidized by m-chloroperoxybenzoic acid (mCPBA) in dichloromethane to obtain title compounds 4a-4o (containing phenyltetrazole), 5a-5o (containing phenyldioxazole), 6a-6o (containing 4-methoxylphenyltetrazole), and 7a-7o (containing 4-chlorophenyldioxazole) [37]. 1 H NMR, 13 C NMR spectra and HRMS spectra of all compounds are shown in the experimental section and Supplementary Materials.

Evaluation of Inhibition of P. aeruginosa Biofilm and Structure-Activity Relationship (SAR) Studies
First, all target compounds were tested for their inhibitory activities against P. aeruginosa PAO1 biofilms (Table 1); 2-aminobenzimidazole was set as the positive control [38]. On the basis of previous studies, phenyldioxazole or phenyltetrazole sulfoxide derivatives were preferred for study. As shown in Table 1, in contrast to the good biofilm inhibitory activity in 5a-5o, there was little or no effect observed in the 4a-4o series compounds. We observed that the biofilm activity of most compounds in series 5 was higher than that of the positive control, which may be due to the introduction of five-membered rings by oxygen atoms. Among them, 5b (R 2 was substituted by para-chloro, inhibition rate was 46.85 ± 2.76%) and 5f (R 2 was substituted by 4-naphthyl; inhibition rate was 40.88 ± 0.75%) had the most significant inhibitory activity. However, the activities of 5c and 5d were Molecules 2023, 28, 3879 4 of 19 weaker than those of 5b (5c, 23.26 ± 3.20%; 5d, 17.55 ± 2.85%), indicating that substitution of the para-position of the benzene ring may have better inhibitory activity. In addition, when R 2 was substituted at the para-position of the benzene ring, the inhibitory activity of chlorinated derivative was higher than that of fluorinated derivative (5h, 23.17 ± 1.10%) and brominated derivative (5i, 21.94 ± 2.72%). When R 2 were alkyl-branched chains, 5n and 5o showed no inhibitory activity (5n, 0.45 ± 0.19%; 5o, 4.75 ± 0.96%), indicating that the aromatic ring was a necessary active group. Based on preliminary SAR analysis, only the para-substitution on the R 2 benzene ring was conducive to activity; in particular, the para-chloro group promoted the activity. It should be noted that the three aryl groups were the optimal framework for designing phenyloxazole inhibitors.

Evaluation of Inhibition of P. aeruginosa Biofilm and Structure-Activity Relationship (SAR) Studies
First, all target compounds were tested for their inhibitory activities against P. aeruginosa PAO1 biofilms (Table 1); 2-aminobenzimidazole was set as the positive control [38]. On the basis of previous studies, phenyldioxazole or phenyltetrazole sulfoxide derivatives were preferred for study. As shown in Table 1, in contrast to the good biofilm inhibitory activity in 5a-5o, there was little or no effect observed in the 4a-4o series compounds. We observed that the biofilm activity of most compounds in series 5 was higher than that of the positive control, which may be due to the introduction of five-membered rings by oxygen atoms. Among them, 5b (R 2 was substituted by para-chloro, inhibition rate was 46.85 ± 2.76%) and 5f (R 2 was substituted by 4-naphthyl; inhibition rate was 40.88 ± 0.75%) had the most significant inhibitory activity. However, the activities of 5c and 5d were weaker than those of 5b (5c, 23.26 ± 3.20%; 5d, 17.55 ± 2.85%), indicating that substitution of the para-position of the benzene ring may have better inhibitory activity. In addition, when R 2 was substituted at the para-position of the benzene ring, the inhibitory activity of chlorinated derivative was higher than that of fluorinated derivative (5h, 23.17 ± 1.10%) and brominated derivative (5i, 21.94 ± 2.72%). When R 2 were alkyl-branched chains, 5n and 5o showed no inhibitory activity (5n, 0.45 ± 0.19%; 5o, 4.75 ± 0.96%), indicating that the aromatic ring was a necessary active group. Based on preliminary SAR analysis, only the parasubstitution on the R 2 benzene ring was conducive to activity; in particular, the para-chloro group promoted the activity. It should be noted that the three aryl groups were the optimal framework for designing phenyloxazole inhibitors. Scheme 1. The synthesis route of the title compounds. Compared with the 4a-4o series, the anti-biofilm activity was not significantly improved by introducing the R 1 as para-methoxy group in 6a-6o series derivatives. In the 7a-7o series, the antibiofilm activity of replacing R 1 with para-chloro substitution was not as good as that of the 5a-5o series. In conclusion, when the para-chloro-substituted phenyloxazole ring and para-methoxyl-substituted phenyltetrazole were applied, the inhibitory activity against P. aeruginosa biofilm was not improved. These experimental results showed that the para-chloro substitution group was not essential for activity as R 1 on the benzene ring compared to chloro as the R 2 on the other side. In addition, we also tested the P. aeruginosa biofilm inhibitory activity of the corresponding intermediate thioethers of 5b and 5f (the biofilm inhibition rate of the intermediate thioether of 5b was −3.77 ± 1.16%, and 5f intermediate thioether was −5.04 ± 0.52%). These results revealed that the thioether derivatives of phenyloxazole had no PAO1 biofilm inhibitory activity, and they showed inhibitory activity against PAO1 biofilms only after being oxidized as sulfoxides. Therefore, the sulfoxide group was verified as an essential active functional group for inhibiting biofilm formation.

Effect of Sulfoxide Derivatives on QS System Reporter Strains
As previously mentioned, las, rhl, and pqs pathways are critical for the regulation of biofilm formation and the secretion of virulence factors in the QS system of P. aeruginosa. According to the regulatory process of the QS system in P. aeruginosa, Givskov et al. [18,39,40] reported the promoters in the QS pathway, including the lasB gene encoding elastase, which was shown to be under the transcriptional control of LasR; the first gene rhlA encodes the rhl operon of rhamnotransferase, and the first gene pqsA encodes the pqsABCDE of the PQS molecule. They were respectively fused with unstable green fluorescent protein (GFP) to construct three reporter strains, PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp, and a detection system for screening small molecule QSIs was established [41,42].
To further explore the mechanism of biofilm formation inhibition caused by the synthetic compounds, reporter strains PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp were introduced. Five compounds, 5a, 5b, 5f, 5j, and 5k, which showed the best antibiofilm activities, were selected for study ( Figure 2). The experimental results verified that at the concentration of 20 µM, all five compounds could inhibit the fluorescence expression of the PAO1-lasB-gfp strain; of these compounds 5b and 5f had the best inhibitory effect ( Figure 2A). Although 5a, 5b, 5f, 5k, and 5j had certain inhibitory effects on the PAO1-lasBgfp and PAO1-pqsA-gfp strains, the expression of rhlA-gfp was not particularly affected under the same experimental conditions ( Figure 2B,C). The las system is upstream of the quorum sensing network [17], and biological reporter gene analysis indicated that phenyloxadiazole sulfoxide derivatives may specifically exhibited anti-biofilm activity via the las pathway. To further explore the mechanism of biofilm formation inhibition caused by the synthetic compounds, reporter strains PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp were introduced. Five compounds, 5a, 5b, 5f, 5j, and 5k, which showed the best anti-biofilm activities, were selected for study ( Figure 2). The experimental results verified that at the concentration of 20 µM, all five compounds could inhibit the fluorescence expression of the PAO1-lasB-gfp strain; of these compounds 5b and 5f had the best inhibitory effect ( Figure 2A). Although 5a, 5b, 5f, 5k, and 5j had certain inhibitory effects on the PAO1-lasB-gfp and PAO1-pqsA-gfp strains, the expression of rhlA-gfp was not particularly affected under the same experimental conditions ( Figure 2B,C). The las system is upstream of the quorum sensing network [17], and biological reporter gene analysis indicated that phenyloxadiazole sulfoxide derivatives may specifically exhibited anti-biofilm activity via the las pathway.

The Effect of 5b on PAO1-lasB-gfp and P. aeruginosa PAO1 Biofilm Growth and Formation
Based on the experimental results, 5b was selected as the key research object. ( Figure  3). Under the premise of no effect on the growth function of the PAO1-lasB-gfp reporter strain ( Figure 3A), 5b showed dose-dependent fluorescence inhibition of the PAO1-lasBgfp strain at different concentrations of 20 µM, 10 µM, 5 µM, 2.5 µM, and 1.25 ( Figure 3B).

The Effect of 5b on PAO1-lasB-gfp and P. aeruginosa PAO1 Biofilm Growth and Formation
Based on the experimental results, 5b was selected as the key research object. (Figure 3). Under the premise of no effect on the growth function of the PAO1-lasB-gfp reporter strain ( Figure 3A), 5b showed dose-dependent fluorescence inhibition of the PAO1-lasB-gfp strain at different concentrations of 20 µM, 10 µM, 5 µM, 2.5 µM, and 1.25 ( Figure 3B). Based on the dose-response curves obtained, the IC 50 value of 5b against the PAO1-lasB-gfp strain was calculated as 3.53 ± 0.16 µM ( Figure 3C). Similarly, we further verified the effect of 5b on the growth and formation of the PAO1 biofilm. The effect of 5b on the growth of P. aeruginosa PAO1 was assessed by monitoring the OD 600 of the culture hourly. The results showed that the normal growth of P. aeruginosa PAO1 was not affected when the maximum concentration of 5b was 50 µM ( Figure 3D). Compound 5b was incubated at concentrations of 50, 25, 12.5, 5, and 2.5 µM, and the control for 24 h and the OD values at 600 nm were evaluated before the biofilm experiments. We found that 5b could reduce the biofilm formation in a dose-dependent manner ( Figure 3E). In addition, the reduction of biofilm formation by 5b was also observed by confocal laser scanning microscopy (CLSM) ( Figure 3F). As shown, biofilm formed with 50 µM of 5b was shallower than the control biofilm; the height of the biofilm formed with the control group, positive group, and 5b were 35, 27, and 14 µm, respectively.

Effect of 5b on Virulence Factors
Further, 5b was used as a template to demonstrate that it inhibits biofilm formation through the las pathway of P. aeruginosa. We measured the effect of 5b on the production of three virulence factors, elastase, pyocyanin, and rhamnolipid, in P. aeruginosa PAO1, which were regulated by las, rhl, and pqs systems, respectively. The results showed that 5b could reduce elastase production in a concentration-dependent manner at a concentration of 50 µM, 25 µM, and 12.5 µM ( Figure 4A). Compound 5b inhibited pyocyanin production only at high concentrations and had little effect on rhamnolipid production. In summary, 5b suppressed the expression of the QS system lasB gene and decreased the production of the virulence factor elastase. Thus, 5b inhibited the formation of the PAO1 biofilm through the las pathway.

Molecular Docking Study
Furthermore, we explored the binding properties of 5b and QS-associated proteins by molecular docking. As the autoinducer of the las pathway in P. aeruginosa, OdDHL binds to the cytoplasmic receptor protein LasR after synthesis by the LasI protein and forms the complex LasR-OdDHL, which binds to the promoter region of the target gene. The LasR-OdDHL complex then induces gene transcription of various virulence factors and specific proteins [43]. Silico molecular docking was performed to predict the binding models of 5b and 5f to the homologous signal receptor protein LasR ( Figure 5, Table 2). The lowest binding energies of the docked conformations were selected from 30 hypothetical conformations as the modes for the corresponding compounds. The docking results are shown in Figure 5, where the benzene ring in the phenyloxazole structure of compound 5f forms hydrophobic interactions with the residue Gly 126; the π bond in the naphthalene ring also developed hydrophobic interaction with the key residues Tyr 56, while the π bond in the chlorophenyl ring of 5b had strong hydrophobic interactions with Trp 88 and Phe 101. It should be noted that the para-chlorinated benzene ring also formed binding interactions with the residue Leu 110, which demonstrates the importance of the para-chlorinated characteristics in phenyloxazole sulfoxide derivatives for P. aeruginosa biofilm activity. These results are consistent with the SAR results observed above and further interpret the active mechanism of phenyloxazole derivatives from the perspective of target and protein binding.

Molecular Docking Study
Furthermore, we explored the binding properties of 5b and QS-associated proteins by molecular docking. As the autoinducer of the las pathway in P. aeruginosa, OdDHL binds to the cytoplasmic receptor protein LasR after synthesis by the LasI protein and forms the complex LasR-OdDHL, which binds to the promoter region of the target gene. The LasR-OdDHL complex then induces gene transcription of various virulence factors and specific proteins [43]. Silico molecular docking was performed to predict the binding models of 5b and 5f to the homologous signal receptor protein LasR ( Figure 5, Table 2). The lowest binding energies of the docked conformations were selected from 30 hypothetical conformations as the modes for the corresponding compounds. The docking results are shown in Figure 5, where the benzene ring in the phenyloxazole structure of compound 5f forms hydrophobic interactions with the residue Gly 126; the π bond in the naphthalene ring also developed hydrophobic interaction with the key residues Tyr 56, while the π bond in the chlorophenyl ring of 5b had strong hydrophobic interactions with Trp 88 and Phe 101. It should be noted that the para-chlorinated benzene ring also formed binding interactions with the residue Leu 110, which demonstrates the importance of the parachlorinated characteristics in phenyloxazole sulfoxide derivatives for P. aeruginosa biofilm activity. These results are consistent with the SAR results observed above and further interpret the active mechanism of phenyloxazole derivatives from the perspective of target and protein binding.
Trp 88 and Phe 101. It should be noted that the para-chlorinated benzene ring also formed binding interactions with the residue Leu 110, which demonstrates the importance of the para-chlorinated characteristics in phenyloxazole sulfoxide derivatives for P. aeruginosa biofilm activity. These results are consistent with the SAR results observed above and further interpret the active mechanism of phenyloxazole derivatives from the perspective of target and protein binding.   All solvents and reagents were obtained from commercial sources without further purification. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 400 at 600 and 150 MHz or 400 and 100 MHz spectrometer. Chemical shifts were recorded as δ in units of parts per million (ppm), while tetramethylsilane (TMS) was used as an internal standard. Compounds were dissolved in CDCl 3 . Mass spectra were recorded on a SCIEX series X500B QTOF mass spectrometer. Thin-layer chromatography (TLC) was performed using Huanghai GF254 Silica gel plates. Column chromatography was performed using silica gel (200-300 mesh, Beijing, China) with a linear solvent gradient.

General Synthesis Method for Compounds 4a-7o
To a solution of heterocyclic thiol (2 mmol) and Et 3 N (3 mmol) dropwise in MeCN (10 mL) were added benzyl bromide (2.4 mmol). The mixture was stirred for 2-4 h at room temperature (monitored by TLC) and quenched with 6M HCl aqueous. The mixture was then extracted by EtOAc (10 mL × 3), saturated in aqueous brine (5 mL × 2), dried over Na 2 SO 4 , and concentrated to dryness. The crude residue was purified by silica gel column, followed by gradient elution with a petroleum ether/ethyl acetate mixture (50/1-30/1 ratio) to provide intermediate thioethers. Thioethers intermediate (1 mmol) and m-chloroperoxybenzoic acid (1 mmol) were added to a solvent of CH 2 Cl 2 (5 mL). The reaction mixture was stirred for 2-4 h at room temperature (monitored by TLC), and then washed with saturated aqueous NaHCO 3 (5 mL × 2) and brine (5 mL × 2), dried over Na 2 SO 4 , and concentrated to dryness. The residue was directly loaded onto a silica gel column followed by gradient elution with petroleum ether/ethyl acetate mixture (30/1-10/1 ratio) to obtain target compounds (4a-4o, 5a-5o, 6a-6o, 7a-7o). the microtiter plate to reach a final inhibitor concentration of 20 µM. DMSO control with 0.2% final concentration was used. The microtiter plate was incubated in a Molecular Devices SpectraMax microplate reader at 37 • C, with GFP fluorescence signals (excitation 485 nm, emission 535 nm) and cell density (OD 600 ) measured every 20 min for at least 12 h. The Inhibition assay of all test compounds and controls were determined in triplicate. P. aeruginosa Rhl and Pqs inhibition assays were performed using a similar method to that of the LasB inhibition assay.

CLSM Images
P. aeruginosa PAO1 was cultured overnight and diluted 100-fold, then the test compounds and bacterial suspension were added to the plate, followed by incubation at 37 • C for 24 h. Floating bacteria were poured out and washed with water three times, then fixed with 4% paraformaldehyde for 15 min and stained with 0.01% acridine orange for 15 min in the dark; excess dye was then washed with PBS. The established model was observed by confocal laser scanning microscope (Nikon-Eclipse-Ti) under green fluorescence light (excitation wavelength: 488 nm, emission wavelength: 515 nm). The signal was received by the FITC channel, where the objective lens was ×10, and scanned layer by layer along the Z-axis from outside to inside.

Quantification Analysis of Elastase
The elastase quantification assay was performed as previously described [46]. An overnight culture of P. aeruginosa PAO1 was diluted to a density of OD 600 = 0.01 in LB medium and inoculated with compounds in a 20 mL conical flask, then incubated at 37 • C with shaking at 200 rpm for 24 h. The cultures were centrifuged at 10,000 rpm at 4 • C for 10 min, and the supernatant was collected and filtered with a 0.22 mm-pore size filter. A supernatant fraction of 100 µL was added to 900 µL of Elastin-Congo Red reaction buffer (2 mg/mL ECR, 0.1 mM Tris-HCl), which was shaken at 37 • C for 18 h. The reaction was placed on ice and 100 µL of 0.12 M EDTA was added to terminate the reaction, before being centrifuged at 4 • C, 12,000 r/min for 10 min; the supernatant was measured at 495 nm.

Determination of Pyocyanin Production
The pyocyanin quantification assay is based on the absorbance of pyocyanin at 520 nm in acidic solution [47]. P. aeruginosa PAO1 was cultured overnight and diluted to OD 600 = 0.01 in LB medium, and then inoculated with compounds in a 20 mL conical flask before being incubated at 37 • C, with shaking at 200 rpm for 24 h. The bacterial culture was centrifuged at 10,000 rpm for 10 min; the supernatant was collected and extracted with chloroform, then to the chloroform layer was added 0.2 M HCl for extraction (after the hydrochloric acid mixed reaction turned pink). The absorbance of the HCl layer was measured at 520 nm by microplate reader.

Detection of Rhamnolipid Production
Rhamnolipid production was directly quantified using the orcinol assay according to the original protocol by Koch et al. [48]. P. aeruginosa PAO1 overnight culture was diluted 100-fold in LB medium and inoculated with compounds in a 20 mL conical flask. The cultures were incubated for 24 h at 37 • C, under shaking condition (200 rpm). Supernatants were collected after the mixture was centrifuged at 10,000 rpm for 10 min and extracted twice by diethyl ether. The ether fraction was evaporated to dry and then resuspended in deionized water and supplemented with orcinal solution (0.19% (w/v) orcinol, 50% H 2 SO 4 ). The mixture was incubated in a water bath at 80 • C for 30 min, and then cooled at room temperature for 15 min; the OD value at 421 nm was measured.

Molecular Docking
Compounds and OdDHL were drawn with ChemBioDraw Ultra (ver. 13.0) software (Cambridge, MA, USA) and minimized with Molecular Operate Environment software (Innovation Center of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, China). The receptor protein lasR in PDB format was downloaded from the RCSB Protein Data Bank (http://www.pdb.org (accessed on 20 July 2022)). A LasR X-ray crystal structure with 1.80 A • resolution (PDB ID: 2UV0) was used for the docking study [49]. The process of deleting water, adding hydrogen, adding Gasteiger charges, and so on, was prepared by Autodock Tools software (San Diego, CA, USA). The OdDHL binding pocket was selected as the docking site, and the docking environment was set in the solvent. Docking was performed after the setting method (placement: triangular matcher, refinement: rigid receptor), score (placement: London dG, refinement: GBVI/WSA dG), and posture (placement: 30, refinement: 5). The optimal docking posture was selected to analyze the interaction between LasR and the target compounds.

Conclusions
The establishment of biofilms can protect P. aeruginosa and help to elude eradication by human immunity and antibacterial drugs. Clinically, once the biofilm is formed on abiotic or tissue surfaces, it is generally considered a pathogenic characteristic of chronic infection. The difficulty in treating P. aeruginosa infections with antibiotics is that almost all patients with cystic fibrosis eventually contract an incurable resistant strain [3,22]. QSIs that reduce the virulence of pathogens without killing pathogenic bacteria are considered feasible targets for the development of antimicrobial agents against P. aeruginosa. They can also alleviate the pressure of drug resistance to a certain extent. In this study, phenyloxadiazole sulfoxide derivative 5b could inhibit the biofilm formation of P. aeruginosa but had no growth inhibitory effect. Similarly, in phenotypic experiments with virulence factors, the decreased production of extracellular virulence factor elastase was observed. Mechanism research results confirm that 5b can effectively inhibit the las system in a dose-dependent manner, and the IC 50 value of inhibitory concentration against the PAO1-lasB-gfp strain was 3.53 ± 0.16 µM. Molecular docking analysis showed that 5b and LasR receptor proteins inhibited the production of virulence factors and Pseudomonas aeruginosa biofilms by forming hydrogen bonds and hydrophobic interactions. In conclusion, sulfoxide derivatives have been proposed as a new QSIs model, which provides a new strategy for the development of new antimicrobial agents and attenuating the pathogenicity of P. aeruginosa.